The goal of this SBIR project is to design and develop an efficient, physiologically relevant and cost-effective 96-well 3-D Cell Migration Assay that is compatible with mid-high throughput/high content assay platforms. Due to its importance in cancer and associated metastatic processes, cell migration is intensively studied and in vitro cell migration assays are routinely used in drug discovery programs aimed at identifying new cancer therapeutics. Cell migration studies of cells grown in 3-D biomimetic matrices are currently performed primarily using trans-membrane well inserts and microfluidic systems. These methods are expensive, laborious and provide inconsistent data. Moreover, these assays compromise on an important characteristic of the in vivo environment: the unique spatial arrangement of cells that facilitates their ability to exhibit normal physiological phenotypes and functions. A major barrier to efficient screening of candidate cancer therapeutics affecting cell migration is the lack of affordable cell based assays that are robust, physiologically relevant, reproducible and cost-effective to perform. The Oris 3-D migration assay proposed in this Phase I project builds on the successful Oris Pro cell migration assay product that Platypus developed and commercialized with the support of a prior SBIR award. The Oris 3-D assay will allow monitoring of cell migration in a more closely in vivo-like microenvironment, thereby facilitating identification and development of lead therapeutic compounds that target cellular responses. The proposed assay is fully automatable and is compatible with readout by microscopy, multi-well plate readers, and high content imagers. Major additional benefits of the proposed assay include ability to i) provide cells with a fully 3-D microenvironment in the entire duration of the migration assay; (ii) use a universal system for both adherent and non-adherent cells; iii) visualize cell movement in real-time and iv) perform multiplexed secondary screens to elucidate mechanism of action of the test compound via cytostaining within the same well. This latter benefit is an extraordinary advantage that increases the knowledge-generating power of the research dollar by conserving reagents, cells, and other resources that would otherwise be consumed in the repetitive testing required by competing formats.